Synthetic peptides and kits for diagnosis of anti-phospholipid syndrome

ABSTRACT

Synthetic peptides and derivatives thereof capable of inhibiting the biological activity of anti-beta-2-glycoprotein 1 (β2GPI) monoclonal antibodies (mAbs) in vitro, and of inhibiting induction of experimental anti-phospholipid syndrome (APS) in mice by anti-β2GPI mAbs, are provided for the diagnosis and treatment of anti-phospholipid syndrome in humans.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of application Ser. No.09/743,225, filed Jan. 8, 2001 now U.S. Pat. No. 6,825,319, which is a371 national stage application of PCT/IL99/00366, filed Jul. 6, 1999,the entire contents of which are hereby incoporated by reference.Application PCT/IL99/00366 claims priority to Israeli application125262, filed Jul. 7, 1998.

FIELD OF THE INVENTION

The present invention relates to synthetic peptides and topharmaceutical compositions comprising them for the diagnosis andtreatment of anti-phospholipid syndrome.

ABBREVIATIONS: AFC: antibody-forming cells; APS: anti-phospholipidsyndrome; HUVEC: human umbilical vein endothelial cells; mAb: monoclonalantibody; MAP: multiple antigenic peptide; PBL: peripheral bloodlymphocytes; SLE: systemic lupus erythematosus; St: streptavidin; β2GPI:beta-2-glycoprotein 1.

BACKGROUND OF THE INVENTION

Autoimmune diseases are disorders in which the immune system producesautoantibodies directed against an endogenous antigen, with consequentinjury to tissues. These self antigens, called also autoantigens,despite being normal tissue constituents, are the target of a humoral orcell-mediated immune response that characterizes the autoimmune disease.

Several connective tissue disorders including vascular diseases, such asvasculitis, systemic lupus erythematosus (SLE), and polymyositis,neurologic diseases such as multiple sclerosis and myasthenia gravis,and hematologic diseases such as idiopathic thrombocytopenia purpura(ITP) and anti-phospholipid syndrome (APS) seem to be caused by anautoimmune reaction. For some of these disorders, the self antigen hasbeen identified and/or pathogenic autoantibodies have been identifiedand isolated.

No specific drugs exist nowadays for the treatment of autoimmunediseases and patients are treated with anti-inflammatory drugs such ascorticosteroids and/or immunosuppressive drugs. All research beingcarried out in this field is directed to the development of drugsspecific for each disease.

Anti-phospholipid antibodies have been associated with a variety ofclinical phenomena, including arterial and venous thrombosis,thrombocytopenia, and obstetric complications. The term“anti-phospholipid syndrome” is used to link a variety of thromboticevents to antibodies against specific proteins involved in bloodcoagulation. Thrombotic events are reported in approximately 30% ofpatients with anti-phospholipid antibodies, with an overall incidence of2.5% patients/year. Deep vein thrombosis of the legs and/or thromboticevents, and cerebral arterial thrombosis are the most common arterialcomplications. Obstetric complications include recurrent spontaneousmiscarriages, fetal deaths, or fetal growth retardations. Women withanti-phospholipid antibodies are particularly prone to second or thirdtrimester fetal death.

The anti-phospholipid syndrome (APS) is characterized by the presence ofhigh titers of anti-cardiolipin and/or anti-β2GPI(beta-2-glycoprotein 1) antibodies which might have lupus anti-coagulantactivity leading to thromboembolic phenomena, thrombocytopenia,recurrent fetal loss, as well as other multisystemic involvements. APScan emerge as a primary syndrome or as secondary syndrome to SLE (Hugheset al., 1986; McNeil et al., 1991).

Anti-β2GPI antibodies bind anionic phospholipids through the β2GPImolecule (McNeil et al., 1990; Igarashi et al., 1996). β2GPI is thetarget antigen for the autoimmune anti-β2GPI antibodies previouslyentitled ‘anti-cardiolipin/anti-phospholipid β2GPI dependentantibodies’. β2GPI (50 KD), initially described by Schultze et al.(1961), is composed of five respective consensus (‘sushi’ like) repeats(Kandiah and Krilis, 1994). β2GPI binds negatively charged phospholipidsthrough a lysine-rich locus (Cys281–Cys288) located in the fifth domain(Hunt and Krilis, 1994) and possesses several in vitro properties whichdefine it as an anticoagulant, i.e., it causes inhibition ofprothrombinase activity, ADP-induced platelet aggregation, plateletfactor IX production (Sheng et al., 1996). Employing site-directedmutagenesis of recombinant human β2GPI, a cluster of lysine residuesthat are critical for phospholipid binding and anti-cardiolipin antibodyactivity was identified (Sheng et al., 1996).

The anti-β2GPI antibodies have been considered to exert a directpathogenic effect by interfering with hemostatic reactions occurring onthe surface of platelets or vascular endothelial cells (Shi et al, 1993;Simantov et al., 1995). Passive transfer of these antibodies into naivemice or mice prone to develop APS, resulted in induction of experimentalAPS in mice (Blank et al., 1991). It has been shown recently (Del Papaet al., 1997; George et al., 1998) that human polyclonal and monoclonalanti-β2GPI antibodies react in vitro with endothelial cells throughadherent β2GPI and induce differential endothelial cell activation. Itis not clear to which epitopes on the β2GPI molecule these anti-β2GPIantibodies are directed, and the correlation to their biologicalactivity.

Attempts have been made to find peptides that could mimic the selfantigen-epitope and would inhibit the autoantibody/self antigen bindingand consequent injury to the tissue. Thus, recently, peptides selectedfrom phage-epitope libraries through binding to pathogenic monoclonalautoantibodies were shown to provide a surrogate antigen or mimotopethat inhibits binding to the original antigen. Such peptides reflect thesequence or conformation of the antigen-binding site, and the finespecificity of the autoantibodies to the protein and non-protein, e.g.polysaccharides or dsDNA, antigens (Scott and Smith, 1990; Scott et al.,1992; Yayon et al., 1993).

SUMMARY OF THE INVENTION

It is an object of the present invention to provide means for diagnosisand specific treatment of the autoimmune disorder anti-phospholipidsyndrome (APS).

A further object of the invention is to provide means for inactivatingB-cells responsible for the production of autoantibodies appearing inAPS patients.

The present invention relates to synthetic peptides suitable for thediagnosis and treatment of APS, more particularly to synthetic peptidesand derivatives thereof capable of inhibiting the biological activity ofanti-β2GPI mAbs in vitro, and of inhibiting induction of experimentalAPS in mice by anti-β2GPI mAbs.

In a preferred embodiment, the peptides of the invention and theirderivatives are selected from the group consisting of:

-   -   (i) a peptide of at least 4 amino acid residues comprising a        sequence selected from:        -   (a) Thr-Pro-Arg-Val (residues 1–4 of SEQ ID NO:1)        -   (b) Lys-Ala-Thr-Phe (residues 3–6 of SEQ ID NO:4)        -   (c) Leu-Arg-Val-Tyr (residues 4–7 of SEQ ID NO:7)    -   (ii) a cyclic derivative of a peptide of (i);    -   (iii) a peptide according to (i) or (ii) in which one or more        amino acid residues have been replaced by the corresponding        D-isomer or by a non-natural amino acid residue;    -   (iv) a chemical derivative of a peptide according to (i)–(iii);    -   (v) a multichain peptide-oligomer/polymer conjugate comprising        two or more of the same or different peptides or peptide        derivatives (i) to (iv) attached to a native or synthetic        multifunctional oligomeric or macromolecular backbone; and    -   (vi) a multiple antigen peptide (MAP) in which two to eight same        or different peptides or peptide derivatives (i) to (iv) are        attached to a diaminoalkanoic acid core.

In one embodiment, a peptide according to (i)(a) above has a sequenceselected from:

-   -   (a1) Leu Lys Thr Pro Arg Val (SEQ ID NO:1    -   (a2) Lys Thr Pro Arg Val Thr (SEQ ID NO:2)    -   (A) Asn Thr Leu Lys Thr Pro Arg Val Gly Gly (SEQ ID NO:3)

In another embodiment, a peptide according to (i)(b) above has asequence selected from:

-   -   (b1) Lys Asp Lys Ala Thr Phe (residues 1–6 of SEQ ID NO:4)    -   (B) Lys Asp Lys Ala Thr Phe Gly Thr His Asp Gly (SEQ ID NO:4)

In a further embodiment, a peptide according to (i)(c) above has asequence selected from:

-   -   (c1) Thr Leu Arg Val Tyr Lys (residues 3–8 of SEQ ID NO:7)    -   (c2) Thr Lys Leu Arg Val Tyr (SEQ ID NO:5)    -   (c3) Thr Leu Leu Arg Val Tyr (SEQ ID NO:6)    -   (C) Cys Ala Thr Leu Arg Val Tyr Lys Gly Gly (SEQ ID NO:7)

Peptides (a1), (a2), (b1), (c1), (c2), and (c3) were obtained using aphage display hexapeptide epitope library and, based on their sequences,peptides A, B, and C were synthesized and shown to neutralize thebiological activity of the anti-β2GPI antibodies and to elicitantiproliferative B-cell response.

It has further been found according to the present invention that when anumber of the same or different peptides or peptide derivatives (i) to(iv) which recognize and bind to autoantibodies secreted by specific Bcells in APS are attached to a multifunctional oligomolecular ormacromolecular backbone, the resulting molecule according to (v) above(hereinafter designated peptide “dimer”, “tetramer”, etc.) is capable ofinhibiting the production of said autoantibodies by said specific Bcells.

The multifunctional oligomolecular or macromolecular backbone may bederived from a native oligomolecular or macromolecular compound such asproteins, oligopeptides, oligosaccharides and oligonucleotides. Suitableproteins are for example albumins, globulins, avidin, and streptavidin.

The multifunctional oligomolecular or macromolecular backbone may alsobe derived from a non-antigenic synthetic oligomer or polymer such asoligolysine, oligoglutamic acid, linear or branched polylysine,polyglutamic acid or copolymers of those containing optionally furtheramino acid residues.

In one preferred embodiment, a peptide or peptide derivative of theinvention is biotinylated and a multichain peptide-protein conjugate isobtained by linking two or four molecules of the same or differentbiotinylated peptide to streptavidin or avidin. According to thisembodiment, “dimers” and “tetramers” of peptides A, B and C wereobtained by attaching 2 or 4 molecules of biotinylated peptides A, B orC, respectively, to one molecule of streptavidin.

The invention further provides multiple antigen peptides (MAPs)according to (vi) above in which two to eight same or different peptidesor peptide derivatives (i) to (iv) are anchored onto a smallimmunogenically inert, two-fold bifurcating diaminoalkanoic acid core.The diaminoalkanoic acid molecule may be ornithine, diaminobutyric acid,homolysine but preferably it is lysine. According to this embodiment,“divalent” and “tetravalent” peptides A, B and C were obtained byattaching 2 or 4 molecules of peptides A, B or C to one or two moleculesof FmocLys(Fmoc)-OH via Gly and/or Ala as spacer.

The invention further provides pharmaceutical compositions comprising apeptide or a derivative thereof of the invention and a pharmaceuticallyacceptable carrier for the treatment of APS, a method for the treatmentof APS which comprises administering to a patient in need thereof aneffective amount of a peptide or peptide derivative of the invention,and a method for inactivating B cells or killing the specific B cellsresponsible for the production of autoantibodies appearing in a patientsuffering from APS which comprises administering to said patient aneffective amount of a multichain peptide or a multiple antigen peptideof the invention.

The invention further provides diagnostic kits comprising one or morepeptides or derivatives thereof of the invention, representing targetepitopes for the diagnosis of anti-phospholipid antibodies withdifferent pathogenic biofunctions, which may correlate either withpregnancy complications, thrombosis, or coagulation dysregulation. Thesekits will allow a quicker diagnosis of the presence of specificautoantibodies, and possibility to provide a more specific treatment forpatients with anti-phospholipid syndrome.

Definitions

In the description hereinafter the following terminology will be used:

Peptide monomer: a single inhibitory peptide A, B, C or irrelevantpeptide D.

Peptide dimer: a conjugate of two molecules of biotinylated peptidemonomer A, B, C, or D, with one molecule of streptavidin, herein St-diA,St-diB, St-diC, or St-diD.

Peptide tetramer: a conjugate of four molecules of biotinylated peptidemonomer A, B, C, or D, with one molecule of streptavidin, hereinSt-tetraA, St-tetraB, St-tetraC, or St-tetraD.

Divalent peptide: an MAP obtained by attaching 2 molecules of peptide A,B, C or D to 2 molecules of FmocLys(Fmoc)-OH via Gly and/or Ala asspacer, wherein the protective Fmoc (9-fluorenylmethyloxycarbonyl) groupis removed during the process of attaching the desired peptide, thusresulting in divalent peptides represented as Lys(α,ε-diA),Lys(α,ε-diB), Lys(α,ε-diC), or Lys(α,ε-diD), herein “divalent A, B, C orD”.

Tetravalent peptide: an MAP obtained by attaching 4 molecules of peptideA, B, C or D to 4 molecules of FmocLys(Fmoc)-OH via Gly and/or Ala asspacer, thus resulting in tetravalent peptides represented as(di-α,ε-Lys) ₂Lys(tetraA), (di-α,ε-Lys)₂Lys(tetraB),(di-α,ε-Lys)₂Lys(tetraC), (di-α,ε-Lys) ₂Lys(tetraD), herein “tetravalentA, B, C or D”.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A–1C are graphs showing inhibition of the anti-β2GPI ILA-1, ILA-3and G-3 mAbs binding to β2GPI by competition assays using increasingconcentrations of the inhibitory monomer peptides A, B and C,respectively, and of unrelated peptide D as control. The percent ofinhibition was calculated as follows: % inhibition=O.D control−O.D withinhibitor/O.D control×100. Each point represents mean±SD of threedifferent experiments.

FIGS. 2A–2C are graphs showing inhibition of the anti-β2GPI ILA-1, ILA-3and G-3 mAbs binding to human umbilical vein endothelial cells (HUVEC)by competition assays using increasing concentrations of the inhibitorymonomer peptides A, B and C, respectively, and of unrelated peptide D ascontrol. The percent of inhibition was calculated as in FIG. 1 above.Each point represents mean±SD of three different experiments.

FIGS. 3A–3C are graphs showing inhibition of U937 monocyte cell adhesionto HUVEC upon exposure to anti-β2GPI ILA-1, ILA-3 and G-3 mAbs andincreasing concentrations of the inhibitory monomer peptides A, B and C,respectively, and of unrelated peptide D as control. The percentage ofinhibition of adhesion was calculated as follows: % inhibition ofadhesion=CPM in the presence of anti-β2GPI mAb−CPM in the presence ofanti-β2GPI mAb and the tested peptide/CPM in the presence of anti-β2GPImAb×100 (CPM=counts/min). Each point represents mean±SD of threedifferent experiments.

FIG. 4 is a bar graph showing inhibition of expression of the adhesionmolecules E-selectin, intracellular adhesion molecule-1 (ICAM-1) andvascular cell adhesion molecule-1 (VCAM-1) on HUVEC by specificinhibitory monomer peptides A, B, or C. To the HUVEC incubated withβ2GPI, were added anti-β2GPI ILA-1, ILA-3 and G-3 mAbs with and withoutpreincubation (2 h) with the peptide monomers A, B and C, respectively,and control peptide D. The expression of adhesion molecules was probedby ELISA employing biotinylated anti-human ICAM-1, VCAM-1, E-selectinand streptavidin-alkaline phosphatase. Each point represents mean±SD ofthree different experiments.

FIG. 5 shows direct binding of sera of APS patients to the inhibitorymonomer peptides A, B or C. ELISA streptavidin-coated plates wereincubated with biotinylated A, B or C, control peptide D or cocktail ofpeptides A+B+C. Following blocking with 5% bovine sera, the patient serawere added at dilution of 1:50. The bound antibodies were detected byadministration of anti-human IgG or IgM alkaline phosphatase andappropriate substrate.

FIGS. 6A–6C show specificity of binding of affinity purified anti-β2GPIIgM and IgG antibodies from APS patients to the inhibitory monomerpeptides A, B and C by ELISA, following preincubation of the testedaffinity purified immuno-globulin with or without the peptide or theβ2GPI molecule, in fluid phase. FIG. 6A: Affinity purified anti-β2GPIIgM and IgG that recognize peptide A were tested for binding to peptideA following preincubation of the purified antibodies with peptide A,cocktail of peptides B+C+D or β2GPI. Each point represents one patient.FIG. 6B: Affinity purified anti-β2GPI IgM and IgG that recognize peptideB were tested for binding to peptide B following preincubation withpeptide B, cocktail of peptides A+C+D or β2GPI. Each point representsone patient. FIG. 6C: Affinity purified anti-β2GPI IgM and IgG thatrecognize peptide C were tested for binding to peptide C followingpreincubation with peptide C, cocktail of peptides A+B+D or β2GPI. Eachpoint represents one patient.

FIG. 7 is a bar graph showing prevention of secretion of anti-β2GPIILA-1, ILA-3, G-3 mAbs by human hybridoma cells secreting them or anirrelevant immunoglobulin herein designated PE, upon exposure to themonomer peptides A, B, C, D, peptide dimers St-diA, St-diB, St-diC,St-diD, and peptide tetramers St-tetraA, St-tetraB, St-tetraC,St-tetraD. The PE hybridoma cells were exposed to a cocktail oftetramers St-tetraA+St-tetraB+St-tetraC. The peptides were used inconcentration of 10 μM.

FIG. 8 is a bar graph showing anti-β2GPI antibody secretion by PBL from2 human APS patients (designated S and Y) in vitro in the presence ofthe inhibitory tetravalent peptides A, B, C(S and Y), A+B (S) andcontrol tetravalent peptide D (S). The peptides were used inconcentration of 10 μM.

FIG. 9 is a bar graph showing the in vitro effect of the inhibitorytetravalent peptides A, B, C, A+B and control tetravalent peptide D onhuman anti-β2GPI antibody-forming cell (AFC) activity from an APSpatient. Enriched B cell population from an APS patient, specific toβ2GPI, were treated with the peptides at concentration of 10 μM for 8 h,washed and incubated overnight at 37° C. in an atmosphere of 7% CO₂. AFCactivity was examined the day after, employing spot ELISA assay.

FIGS. 10 (A–C) show in vitro effect of the inhibitory peptides on humananti-β2GPI AFC activity in an APS patient. Enriched B cell populationfrom an APS patient, specific to β2GPI, was assayed for antibodysecretion following treatment with different concentrations ofinhibitory peptides A, B, C, and control peptide D. Irrelevant anti-DNAAFC were used as negative control. FIG. 10A: monomer A, divalent peptideA, and tetravalent peptides A, D. FIG. 10B: monomer B, divalent peptideB, and tetravalent peptides B, D. FIG. 10C: monomer A+B; divalentpeptidea A+B, tetravalent peptides A+B, D.

FIG. 11 shows the presence of B cell peptide epitopes in mice immunizedwith β2GPI. Sera of immunized mice were screened for the presence ofmouse antibodies to peptide A, B and C, by ELISA, withstreptavidin-coated plates followed by incubation with biotinylatedpeptide A, B, and C. Blocked plates (3% gelatin) were exposed todifferent dilutions of the sera. The binding was probed by anti-mouseIgG alkaline phosphatase.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “peptide derivative” includes a cyclicderivative thereof, an analog in which one or more amino acid residueshave been replaced by the corresponding D-isomer or by a non-naturalamino acid residue, or a chemical derivative thereof.

The term “cyclic peptide” as used herein refers to cyclic derivatives ofa peptide to which two additional amino acid residues suitable forcyclization have been added, one at the carboxyl terminus and one at theamino terminus. Thus, the cyclic peptides contain either anintramolecular disulfide bond, i.e. —S—S—, an intramolecular amide bondbetween the two added residues i.e.—CONH— or —NHCO—, or intramolecularS-alkyl bonds, i.e.—S—(CH₂)_(n)—CONH— or NHCO—(CH₂₎)_(n)—S—, wherein nis 1 or 2.

The cyclic derivatives containing an intramolecular disulfide bond maybe prepared by conventional solid phase synthesis (Merrifield et al.,1982) while incorporating suitable S-protected cysteine or homocysteineresidues at the positions selected for cyclization such as the amino andcarboxyl termini (Sahm et al., 1996). Following completion of the chainassembly, cyclization can be performed either by selective removal ofthe S-protecting groups with a consequent on-support oxidation of freecorresponding two SH-functions, to form S—S bonds, followed byconventional removal of the product from the support and appropriatepurification procedure, or by removal of the peptide from the supportalong with complete side-chain deprotection, followed by oxidation ofthe free SH-functions in highly dilute aqueous solution.

The cyclic derivatives containing an intramolecular amide bond may beprepared by conventional solid phase synthesis while incorporatingsuitable amino and carboxyl side-chain protected amino acid derivativesat the positions selected for cyclization. The cyclic derivativescontaining intramolecular —S-alkyl bonds can be prepared by conventionalsolid phase synthesis while incorporating an amino acid residue with asuitable amino-protected side chain, and a suitable S-protected cysteineor homocysteine residue at the positions selected for cyclization.

A peptide may have one or more of the amino acid residues replaced bythe corresponding D-amino acid residue. Thus the peptide or peptidederivative may be all-L, all-D or a D,L-peptide. In another embodiment,an amino acid residue may be replaced by a non-natural amino acidresidue. Examples of non-naturally occurring amino acids includeNα-methyl amino acids, Cα-methyl amino acids, β-methyl amino acids andamino acid analogs in general such as, but not being limited to,β-alanine (β-Ala), norvaline (Nva), norleucine (Nle), 4-aminobutyricacid (γ-Abu), 2-aminoisobutyric acid (Aib), 6-aminohexanoic acid(ε-Ahx), ornithine (Orn), hydroxyproline (Hyp), sarcosine, citrulline,cysteic acid, and cyclohexylalanine.

A chemical derivative of a peptide includes, but is not limited to, aderivative containing additional chemical moieties not normally a partof the peptide provided that the derivative retains the inhibitoryactivity of the peptide. Examples of such derivatives are: (a) N-acylderivatives of the amino terminal or of another free amino group,wherein the acyl group may be either an alkanoyl group such as acetyl,hexanoyl, octanoyl; an aroyl group, e.g., benzoyl, or biotinyl; (b)esters of the carboxyl terminal or of another free carboxyl or hydroxygroups; and (c) amides of the carboxyl terminal or of another freecarboxyl groups produced by reaction with amonia or with a suitableamine.

The multichain peptide-oligomer/polymer conjugate preferably containstwo or more identical or different peptides or peptide derivativesattached to an oligomeric or polymeric backbone wherein each of saidpeptides was identified first to bind to an autoantibody derived from anAPS patient and to inhibit the activity of said autoantibody in vitroand in vivo in experimental animals. A multichain peptide conjugatecontaining a number of different peptides might be preferred.

The peptide or peptide derivative residues can be attached to anoligomer or polymer backbone by any suitable known procedure such as bychemical coupling of the peptide with a water-soluble carbodiimide, e.g.DCC, and then performing the conjugation with the polymer or oligomer byknown techniques (Muller et al, 1982).

The multiple antigenic peptide (MAP) system is based on a smallimmunogenically inert core molecule of radially branching diaminoalkanoic acid, preferably lysine, dendrites onto which a number ofpeptide antigens are anchored (Tam, 1988 and 1989; Posnett et al.,1989). The thus resulting large macromolecule has an uniquethree-dimensional configuration with a high molar ratio of peptideantigen to core molecule. The MAPs are prepared by standard solid-phasepeptide synthesis whereby the inert MAP core, e.g. lysine, is attachedto a solid-phase peptide synthesis support, preferably via a spacer, andthe desired peptide antigens are synthesized directly on thebranched-lysine core. After the synthesis is complete the MAPmacromolecule is cleaved from the support using standard techniques. MAPcore molecules attached to several different resin supports arecommercially available for use in most automated peptide synthesizers.Both Boc- and Fmoc-strategies can be employed with little or novariation of the standard protocols. The crude cleavage products can beobtained by desalting using a Sephadex column.

The spacer in the MAP can be any aminocarboxylic acid including, but notbeing limited to, aminohexanoic acid and amino acids such as glycine andalanine, or peptides.

According to this embodiment MAPs have been prepared according to theinvention with one lysine core onto which two peptide molecules wereanchored (herein “divalent” peptides) and with two-lysine core ontowhich four peptide molecules were anchored (herein “tetravalent”peptides), and the spacer was alanine.

Examples of divalent peptides with peptides derived from monopeptidesA–C of the invention and irrelevant peptide D are as follows:

-   -   A: Asn Thr Leu Lys Thr Pro Arg Val Gly Gly X Ala (SEQ ID NO:8)    -   B: Lys Asp Lys Ala Thr Phe Gly Thr His Asp Gly Gly X Ala (SEQ ID        NO:9)    -   C: Cys Ala Thr Leu Arg Val Tyr Lys Gly Gly Gly X Ala (SEQ ID        NO:10)    -   D: Pro Val Arg Ser Pro His Gln Ser Tyr Pro Gly Gly Gly X Ala        (SEQ ID NO:11)        wherein X=FmocLys(Fmoc)-OH

Examples of tetravalent peptides with peptides derived from monopeptidesA–C of the invention and irrelevant peptide D are as above except for anadditional X residue between the X and the Ala residues at theC-terminus.

The peptides of the invention, including the multichain and themultivalent peptides, will be given to patients in a form that insurestheir bioavailability, making them suitable for treatment. If more thanone peptide is found to have significantly inhibitory activity, thesepeptides will be given to patients in a formulation containing a mixtureof the peptides or different peptides are attached to the oligomer orpolymer backbone or are anchored onto to the (poly)lysine core.

The invention further includes pharmaceutical compositions comprising atleast one synthetic peptide, a derivative thereof, a multichain ormultiple antigen peptide according to the invention optionally with apharmaceutically acceptable carrier.

Any suitable route of administration is encompassed by the invention,including oral, intravenous, subcutaneous, intraarticular,intramuscular, inhalation, intranasal, intrathecal, intraperitoneal,intradermal, transdermal, or other known routes, including the enteralroute.

The dose ranges for the administration of the compositions of thepresent invention should be large enough to produce the desired effect,whereby, for example, production of the pathogenic autoantibodies andtheir biologic activity are substantially prevented or inhibited, andfurther, where the disease is significantly treated. The doses shouldnot be so large as to cause adverse side effects, such as unwanted crossreactions, generalized immunosuppression, anaphylactic reactions and thelike. The dosage administered will be dependent upon the age, sex,health, and weight of the recipient, kind of concurrent treatment, ifany, frequency of treatment, and the nature of the effect desired.

The invention further provides diagnostic kits comprising one or morepeptides of the invention and, possibly, other anti-β2GPI mAb inhibitorypeptides. Detection of the autoantibodies in serum of the patients maybe carried out with the biotinylated peptides using streptavidin-coatedELISA plates, for example, as described in Example 6 herein.

The invention will now be illustrated by the following non-limitativeExamples.

EXAMPLES

Materials and Methods

(a) Epitope Library. The hexapeptide epitope library was kindly providedby George P. Smith (University of Missouri, Columbia, MO) and wasconstructed by use of the phage fd-derived vector fUSE5 as described(Scott and Smith, 1990). This library consists of 2×10⁸ original phageclones each containing a hexapeptide fused to the minor coat proteinPIII (Scott and Smith, 1990).(b) Anti-β2GPI mAbs. The human anti-β2GPI mAbs named ILA-1, ILA-3 andG-3 (IgM), were prepared by human-human hybridoma technique from PBL ofan APS patient as described (George et al., 1998). Briefly, peripheralblood cells (PBL) were separated from whole blood by Ficoll-Hypaquegradient, the PBL were exposed to pokeweed mitogen for 5 days in orderto enrich the B cell population and the lymphocytes were fused with thehuman lymphoblastoid cell line GM4672 in the presence ofpolyethyleneglycol (PEG 1500). After fusion, the cells were seeded into96-well tissue culture plates with RPMI 1640, 10% FCS andhypoxanthine-aminopterin-thymidine (HAT) selection media. New cloneswere detected after 4–5 weeks, grown and screened for binding to β2GPIby ELISA. All clones were subjected 4 times to limiting dilution cloningprocedures in regular medium. All of the anti-β2GPI mAbs were found toactivate endothelial cells in vitro and to induce experimental APS bypassive transfer (George et al., 1998).(c) Identification of peptides which bind specifically to the anti-β2GPImAbs. The hexapeptide epitope library of section (a) above was used asdescribed (Scott and Smith, 1990). Briefly, a library sample containing3.8×10⁹ infectious phage particles was subjected to three rounds ofselection (panning) and amplification. For each selection cycle abiotinylated mAb prepared as described in section (b) above (10 μg inthe first panning and 1 μg for the others) was added in a total volumeof 50 μl. The phages were preincubated with the biotinylated mAbovernight at 4° C., and the reaction mixtures were then layered in 1 mlPBS on streptavidin-coated 30-mm polystyrene petri dishes (Nunc,Kamstrup, Roskilde, Denmark) for 30 min at room temperature. Unboundphages were removed by extensive washings in PBS, and the remainingphages were eluted with 0.1M glycine-HCl, pH 2.2, and neutralized withTris 1M. Eluted phages were amplified in Escherichia coli K91 and usedas input in the subsequent round of selection. After three rounds ofpanning, individual bacterial colonies containing amplified phage cloneswere grown in microtiter plates overnight at 37° C., and the phages weretested by ELISA for their ability to specifically bind the mAb.

(c)(i) Anti-β2GPI mAb Binding to the Isolated Phages: ELISA plates(Maxisorb, Nunc, Kamstrup, Roskilde, Denmark) were coated with affinitypurified rabbit anti-phage M13 (10 μg/ml 0.1M NaHCO₃, pH 8.6). Followingblocking with 1% gelatin, enriched phage clones, containing 10⁹ phageparticles, were then added to the wells and incubated for 1 hour at 37°C. Wells were blocked with 1% gelatin and incubated with theinvestigated anti-β2GPI mAb (1 μg/ml) overnight at 4° C. The binding ofthe antibody to the immobilized phage was probed with streptavidinalkaline phosphatase (Jackson Immunoresearch Laboratories Inc, WestGrove, Pa., US), and appropriate substrate at O.D. 405 nm. Between eachstep extensive washings were performed.

(c)(ii) Sequencing: Positive phage clones were propagated and their DNAwas sequenced in the epitope region by using a Sequenase version 2.0 kit(United States Biochemical) and the fUSE sequencing primer according tothe manufacturer's instructions.

(d) Peptide Synthesis:

(d)(i) General peptide synthesis. All protected amino acids, couplingreagents and polymers were obtained from either Novabiochem AG(Läufelfingen, Switzerland) or Sygena Ltd. (Liestal, Switzerland).Synthesis grade solvents were obtained from Labscan (Dublin, Ireland).Peptides were prepared by conventional solid phase peptide synthesis,using an ABIMED AMS-422 automated solid-phase multiple peptidesynthesizer (Langenfeld, Germany). The 9-fluorenylmethoxy-carbonyl(Fmoc) strategy was used throughout peptide chain assembly, followingthe company's commercial protocols. In each reaction vessel, Wang resin,which contained the first, covalently bound, corresponding N-FmocC-terminal amino acid (12.5 μmol) was used (typically, polymer loadingof 0.3–0.7 mmol/g resin were employed). Side chain protecting groupswere: tert-butyloxycarbonyl (t-Boc) for Lys, tert-butyl-ester (O-t-But)for Asp, tert-butyl-ether (t-But) for Tyr. Ser and Thr, Trityl (Trt) forAsn Gln, His and Cys, and 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl (PbF) for Arg. Each coupling reaction wasperformed twice, as a rule, using two successive reactions with 50 μmol(4 eqv) of corresponding N-Fmoc-protected amino acid, 50 μmol (4 eqv) ofbenzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBop) reagent, and 100μmol (8 eqv) of 4-N-methyl-morpholine (NMM), alldissolved in dimethyl-formamide (DMF), typically for 20–45 min at roomtemperature. Cleavage of the peptide from the polymer was achieved byreacting the resin with trifluoroacetic acid (TFA)/H₂O/triethylsilane(TES); 90:5:5; v/v, for 2 hours at room temperature. The cleavagemixtures were cooled at 4° C., the peptides were precipitated withice-cold di-tert-butylether (DTBE) and centrifuged for 15 min., 3000 rpmat 4° C. The pellet was washed and centrifuged 3× with DTBE, dissolvedin 30% acetonitrile in H₂O and lyophilized. The product obtained fromeach column was analyzed by HPLC. Following analysis, the products werecombined, redissolved in 30% acetonitrile in H₂O and lyophilized.

(d)(ii) Synthesis of multiple antigenic peptides (MAP). The synthesis ofdivalent and tetravalent derivatives of peptides A, B, C and D wasperformed using an ABIMED AMS-42 synthesizer following exactly theprotocols described above in section (d)(i). Peptide chain assemblystarted from Fmoc-Ala-Wang resin. For the preparation of divalentpeptides, the second coupling step was achieved with N^(α)₁N^(ε)-Fmoc₂-Lys. Removal of Fmoc-protection yielded two amino functionsthat were extended, each, by two or three Gly residues, according to thedesired sequence, and then by the corresponding requisite Fmoc-aminoacids. For the preparation of tetravalent peptides, the third couplingstep was performed with N^(α) ₁, N^(ε)-Fmoc₂-Lys followed by removal ofFmoc and exposure of four amino functions available for chain extension.Processing of peptide-polymer conjugates and isolation of products wereexactly as described above in section (d)(i).

(d)(iii) Biotinylation of peptides and conjugation to streptavidin.Resin-bound peptide (Wang-resin, Calbiochem-Novabiochem AG;Läufelfingen, Switzerland), 11 mg, was suspended in a minimal volume ofN-methyl-2-pyrrolidone (NMP). Biotin-N-hydroxysuccinimide (Sigma), 15μmol, and 15 μmol diisopropylethylamine were added. After 16 hours, thereaction products were washed with NMP, methanol, and ether. Thebiotinylated peptide was deprotected and cleaved from the resin with acleavage mixture containing 5% triethylsilan (Fluka Chemicals, Buchs,Switzerland), 5% water, and 90% trifluoroacetic acid. The cleavedpeptide was precipitated with ice-cold peroxide-free ether and thepellet was dissolved in water and subsequently lyophilized. The degreeof biotinylation was estimated by HPLC and by an optical test based onbinding of 2-(4-hydroxyazobenzene) benzoic acid to biotin (Green, 1965).Yields are usually in the range of 40–90%. Purification of biotinylatedpeptides was achieved by HPLC.

Conjugation to streptavidin: equal volumes of 50 μM streptavidinreconstituted in RPMI and 200 μM biotinylated peptide monomers weremixed at room temperature over 30 min, thus producing a tetramer of fourpeptide molecules conjugated to a streptavidin molecule.

(d)(iv) Reversed phase HPLC: Analysis of synthetic peptides. For puritydetermination, analytical reversed-phase HLPC was performed using aprepacked Lichrospher-100 RP-18 column (4×25 mm, 5 μm bead size)employing a binary gradient formed from 0.1% TFA in H₂O and with 0.1%TFA in 75% acetonitrile in H₂O. All the analyses were performed using aSpectra-Physics SP8800 liquid chromatography system equipped with anApplied Biosystems 757 variable wavelength absorbance detector. Thecolumn effluents were monitored by UV absorbance at 220 nm andchromatograms were recorded on a Chrome-Jet integrator. All solvents andHPLC columns were obtained from Merck (Darmstadt, Germany).

(d)(v) HPLC purification of crude peptides was achieved employingsemi-preparative column (Merck; 250×10 mm; 7 μm) with the abovegradient. The following peptides were synthesized: peptides A, B, C andD as monomers, dimers and tetramers thereof with streptavidin, anddivalent and tetravalent MAPs thereof with Lys.

(d)(vi) Amino acid composition analysis. Peptide solutions wereroto-evaporated (about 40 μg of peptide in 40 μl solution with 5 μg ofnorvaline as an unnatural amino acid internal standard), hydrolyzed in6N HCl at 110° C. for 22 hours under vacuum and analyzed with a HP 1090amino acid analyzer, using on-line pre-column ortho-phthalaldehyde(OPA)/Fmoc derivatization combined with reversed-plase chromatography.This quantification was used as a basis for determining the totalpeptide yield.

(e) Inhibition of anti-β2GPI mAb binding to β2GPI by the inhibitorypeptides. For competition ELISA tests, the anti-β2GPI mAbs (10 μg/ml)were preincubated (overnight at 4° C.) with varying concentrations ofthe peptides. The competition reaction was then transferred toβ2GPI-coated γ-irradiated ELISA plates (Nunc), and the assay continuedas described above. The percentage of inhibition was calculated asfollows: % inhibition=O.D control −O.D with inhibitor/O.D control×100.

(f) Anti-β2GPI mAb binding to endothelial cells in the presence of theinhibitory peptides. Human umbilical vein endothelial cells (HUVEC) wereisolated as previously described and cultured under standard conditions(Jaffe et al., 1973). Briefly, umbilical cords were treated withcollagenase (0.2% from Clostridium histolyticum, Boehringer) for 20 minat 37° C., cells were cultured in medium 199 with 20% FCS, 10 μg/mlgrowth supplement, 100 μg/ml heparin and antibiotics (streptomycin,penicillin), and used at passages 1–2 for plating onto gelatin-coated96-well plates. Cyto-ELISA was performed as detailed before (Jaffe etal., 1973). Briefly, mAbs at different concentrations were added toHUVEC confluent wells, previously coated with 10 μg/ml β2GPI for 2 hoursand fixed with 0.1% glutaraldehyde. The bound mAb was detected byfurther 1-hour incubation with alkaline phosphatase-conjugated goatanti-human IgM.

(g) Adherence of U937 monocyte cells to endothelial cells in thepresence of anti-β2GPI mAbs and the inhibitory peptides. This assay wasperformed as previously described (Carvalho et al., 1996). Briefly, U937cells (a monocyte/macrophage-like cell line) were pretreated withheat-aggregated gamma-globulin for 30 min at 37° C. (to block Fcreceptor binding) and labeled with 0.1 μCi/ml of [³H]-thymidine(Amersham International, Little Chalfont, UK) for 24 hours. Adhesionassays were performed on HUVEC monolayers which were preincubated withβ2GPI and mAbs, with and without different concentrations of specificand irrelevant peptides, overnight. The endothelial cell (EC) monolayerswere extensively washed, and radiolabeled U937 cells were added to eachwell, in RPMI 1640 medium containing 0.2% BSA for 30 min at 37° C. Thenonadherent cells were removed by washing and the cells were lysed withformic acid. Radioactivity associated with adherence was quantified bybeta-scintillation spectroscopy. The results were expressed as percentof added U937 cells that adhered and are presented as the mean±SD from3–5 replicate wells.

(h) ELISA for detecting the expression of adhesion molecules followingexposure to anti-β2GPI mAbs and the inhibitory peptides. HUVEC cellsgrown in 96-well plates and preincubated with mAbs, with and withoutdifferent concentrations of specific and irrelevant peptides (100ug/ml), were washed, fixed with 0.1% glutaraldehyde, and then treatedwith PBS containing 0.2% Triton-X100 in order to permeabilize the cellmembrane (Adamson et al., 1996). The plates were blocked with 3% BSA andincubated with biotinylated mouse anti-human E-selectin, anti-humanintracellular adhesion molecule-1 (ICAM-1) or anti-human vascular celladhesion molecule-1 (VCAM-1) (PharMingen, Torreyana Road, San Diego,Calif.) 1 μg/ml for 1 hour. The cells were then exposed to streptavidinalkaline phosphatase (Jackson) and appropriate substrate.

(i) Mice: Female BALB/c mice, aged 10–12 weeks, were purchased fromTel-Aviv University Animal House, Israel. The females were cagedovernight with BALB/c males and examined for vaginal plug next morning.The presence of the plug was considered as day 0 of pregnancy in all thestudied groups.

(j) Induction of murine experimental APS. Mice were infusedintravenously with 20 μg of each one of the anti-β2GPI ILA-1, ILA-3, andG-3 mAbs at day 0 (the day at which a vaginal plug was observedfollowing mating) (Blank et al., 1991), 6 hours later the mice receiveddaily 40 μg/mouse of the tested peptides during 3 days. The mice werebled and sacrificed on day 15 of pregnancy. Fetal resorptions, activatedpartial thromboplastin time and platelet counts (markers of theAPS-equivalent in mice) were determined as described (Blank et al.,1991).

(k) Anti-β2GPI antibody secretion by hybridoma cells in the presence ofthe inhibitory peptides. Hybridoma cells named ILA-1, ILA-3, and G-3were tested for secretion of anti-β2GPI mAbs following 48 h of treatmentwith specific inhibitory monomer peptides A, B or C, their dimers andtetramers with St, or the corresponding divalent and tetravalentpeptides. PE hybridoma cells secreting irrelevant immunoglobulin wereused as negative control. The hybridoma cultures were washed after 48 hand incubated for 5 more days. Culture fluid was tested for the presenceof anti-β2GPI mAbs by ELISA.

(l) Anti-β2GPI Abs secretion from PBL of APS patients in the presence ofthe inhibitory peptides. PBL separated from APS patient blood onFicoll-Hypaque gradient (Pharmacia) were incubated for 48 h withspecific inhibitory monomer peptides A, B or C, or the correspondingdivalent and tetravalent peptides, at concentration of 10 μM, addeddaily. The PBL cultures were washed 48 h later and incubated for 5 moredays. Culture fluid was tested for the presence of anti-β2GPI IgM andIgG Abs by ELISA.

(m) Detection of human anti-β2GPI Ab secreting B cells in PBL populationfrom APS patient following exposure to the inhibitory peptides. PBL wereseparated on Ficoll-Hypaque gradient (Pharmacia), loaded on an anti-CD19microbead column, followed by streptavidin-microbeads coated withbiotinylated β2GPI. The thus affinity purified B cells were assayed fortheir ability to secrete in vitro anti-β2GPI Abs. The B cells wereincubated for 24 h with the inhibitory peptides A, B or C, or withirrelevant peptide D, as monomers or as divalent and tetravalent MAPs,at different concentrations. The enriched population of total B cells(1×10⁶/ml) were seeded in RPMI 1640 medium in the presence of 10% FCSonto 24-well tissue culture plates (Nunc) precoated with β2GPI (10μg/ml), or DNA (as negative control) (10 μg/ml), and blocked withgelatin. The plates were incubated overnight at 37° C. in an atmosphereof 7% CO₂. Following extensive washings, anti-human IgG or IgMconjugated to alkaline phosphatase was added for 2 h at 37° C. Spots ofimmunoglobulin secreted by each B cell were probed by adding thesubstrate BCIP (Sigma) in 2-aminopropranolol Triton X-405 MgCl₂ bufferto 3% agar (type I, low electroendosis; Sigma) heated and diluted inBCIP buffer at 4:1 ratio, resulting in a 0.6% agar solution. ELISPOTS(blue spots) were evaluated after exposure of the cells to specific andnon-specific peptides.

(n) Statistical analysis: ANOVA statistical analysis was used toevaluate differences between the binding properties and the biologicalactivity of the various studied groups. p<0.05 was considered asstatistically significant.

Example 1

Isolation of Peptide-Presenting Phases Binding Specifically toAnti-β2GPI mAbs

The anti-β2GPI mAbs ILA-1, ILA-3 and G-3 were subjected to a phagelibrary containing random hexapeptides to determine peptide sequenceswhich are recognized by them, as described in Materials and Methods,section (c). Three rounds of selection were performed, and selectedphages were randomly chosen for sequencing after the third round. Thedifferent sequences in phages, probed by the biotinylated mAbs, aresummarized in Table 1.

TABLE 1 Binding of anti-β2GPI mAbs to phage isolated from an hexapeptideepitope library Epitope Binding Phages Antibody sequence* SEQ ID NO:O.D. identified, no. ILA-1 L K T P R V 8 (res. 3–8) 975 ± 72 24 K T P RV T 12 1201 ± 142 18 ILA-3 K D K A T F 13 1178 ± 101 15 G-3 T L R V Y K14 791 ± 33 12 T K L R V Y 15 954 ± 62 7 T L L R V Y 16 869 ± 71 16Binding of anti-β2GPI mAbs 5 μg/ml, to 10⁹ phage particles/well, byELISA (O.D. at 405 nm). Binding of ILA-1 to irrelevant hexapeptidePVRSPH resulted in O.D. of 92 ± 11. *Amino acid sequence (deduced fromDNA sequence) of the inserted hexapeptide.

ILA-1 mAb detected the sequence KTPRV (residues 4–8 of SEQ ID NO:8) thatappeared in 42 clones, wherefrom 24 clones showed the sequence LKTPRV(residues 3–8 of SEQ ID NO:8) which represents a mimotope locatedbetween domain I/II of the β2GPI molecule as LK(C)TPRV (SEQ ID NO:17) onthe native form of the β2GPI. The other 18 clones showed the motifKTPRVT (SEQ ID NO12) presented at the same location on the β2GPI andappearing as K(C)TPRV(CC)T (SEQ ID NO:18).

ILA-3 mAb fished out the linear sequence KDKATF (SEQ ID NO:13) locatedon the fourth domain of the β2GPI molecule (15 clones). ILA-4 mAb probedthe sequence mimotope LVEPWR (SEQ ID NO:19) the location of which onβ2GPI is still undetermined. The anti-β2GPI mAb named G-3 recognized alinear motif sequence LRVY (residues 3–6 of SEQ ID NO:15) located on thethird domain of the β2GPI molecule, that appeared in 37 of the examinedclones.

Example 2

Inhibition of Binding of Anti-β2GPI mAbs to β2GPI and to EndothelialCells by the Synthetic Monomer Peptides A, B and C

(2a) The specificity of interaction of the mAbs with the correspondingsynthetic peptides A, B, C and control peptide D as monomers, wasassessed by inhibition experiments as described in Materials andMethods, sections (e) and (f).

The results depicted in FIG. 1A show that peptide A (filled circles)inhibited ILA-1 mAb binding to β2GPI by 97% at 10 μM peptideconcentration, while peptides B and C, directed to other locations onthe β2GPI molecule, as well as irrelevant peptide D (open circles, opentriangles and open squares, respectively) did not have any inhibitoryeffect (10%, 11% and 8%, respectively), at the same peptideconcentration. Peptide B abrogated the binding of ILA-3 mAb to β2GPI by77% (FIG. 1B, open circles), and 98% inhibition of G-3 mAb binding toβ2GPI was exhibited by peptide C (FIG. 1C, open triangles), both at 10μM peptide concentration. The specificity of the inhibition of bindingof ILA-3 and H-3 mAbs to β2GPI by the tested peptides was clearlyevident when the inhibition assay performed by preincubation of thetested anti-β2GPI mAb with the specific peptide B or C, respectively,was compared to the binding following preincubation with the othertested peptides shown in FIGS. 1B and 1C (peptides A, C, D and A, B, D,respectively).

(2b) The anti-β2GPI mAb binding to HUVEC in the presence of peptides A,B, C and D was carried out as described in Materials and Methods,section (f). The specificity of interaction of the anti-β2GPI mAbscorresponding peptides and HUVEC is shown in FIGS. 2A–C. Peptides A, Band C inhibited the binding of ILA-1, ILA-3 and G-3 mAbs to HUVEC by 95%(FIG. 2A, filled circles), 72% (FIG. 2B, open circles), and 92% (FIG.2C, open triangles), respectively, at peptide concentration of 10 μM. Noinhibition of binding of the anti-β2GPI mAbs to HUVEC by peptidesdirected to other locations on the β2GPI molecule, or by irrelevantpeptide D, was observed (FIGS. 2A–C).

Example 3

Peptides which Bind Specifically to anti-β2GPI mAbs have a PreventiveEffect on the Ability of the anti-β2GPI mAbs to Enhance MonocyteAdhesion to Endothelial Cells

The adhesion of monocytes to endothelial cells (EC) is considered amarker of EC activation. The percentage of adhesion is expressed as theportion of added U937 monocyte cells adhering to the HUVEC (thusreflecting the percentage of HUVEC coverage by the monocytes). Theability of the peptide monomers A, B and C to prevent activation ofHUVEC via decrease of the adhesion percentage of U937 monocytes toHUVEC, was tested as described in Materials and Methods, section (g).

As shown in FIG. 3A, ILA-1 mAb preincubated with peptide A (filledcircles) inhibited the adhesion of U937 to HUVEC under the experimentalconditions used by 84% at 10 μM peptide concentration, as compared to6–17% inhibition in the presence of peptides B and C, or of irrelevantpeptide D (open circles, open triangles and open squares, respectively).The most significant prevention of adhesion of U937 monocyte cells toHUVEC was accomplished by preincubation of G-3 mAb with peptide C with92% inhibition, or by preincubation of ILA-3 mAb with peptide B with 95%inhibition, at peptide concentration of 15 μM (FIGS. 3C and 3B,respectively). The specificity of inhibition of adhesion of U937 cellsto HUVEC by the inhibitory peptides corresponding to each anti-β2GPI mAbwas confirmed, in each case, by using the other peptides specific todifferent anti-β2GPI mAbs or the irrelevant peptide D, as shown in FIGS.3B and 3C (7–21% inhibition, at peptide concentration of 10 μM,p<0.001).

Example 4

Prevention of Expression of the Adhesion Molecules E-Selectin, ICAM-1and VCAM-1 on HUVEC by Peptides Directed Specifically to anti-β2GPImAbs.

The enhancement of monocytes to endothelial cells followed by increasein the amount of adhesion molecules expression, as a result of exposureof endothelial cells to anti-β2GPI mAbs, has been observed previously atthe laboratory of one of the present inventors. The effect of eachspecific peptide monomer A, B, C on the expression of the adhesionmolecules E-selectin, ICAM-1 and VCAM-1 on HUVEC caused by thecorresponding anti-β2GPI mAb, was examined according to Materials andMethods, section (h). The results are shown in FIG. 4, in which theblack columns represent treatment with a specific peptide (i.e.,peptides A, B and C for ILA-1, ILA-3 and G-3 mAbs, respectively), thelined columns represent treatment with a cocktail of non-specificpeptides (i.e., peptides B+C+D, A+C+D and A+B+D for ILA-1, ILA-3 and G-3mAbs, respectively), and the dotted columns correspond topeptide-untreated cells.

The most pronounced inhibitory effect was evident on E-selectinexpression by HUVEC following preincubation of peptide B with ILA-3 mAb(O.D. 0.211±0.064 in comparison to OD 1.591±0.137 in the presence ofirrelevant peptides A+B+C, p<0.001). Peptide B also abrogated theexpression of ICAM-1 (p<0.002) and VCAM-1 (p<0.001) by HUVEC exposed toILA-3 mAb.

Preincubation of the mAb ILA-1 with peptide A or mAb G-3 with peptide Calso resulted in a significant inhibition of E-selectin expression byHUVEC (O.D 0.239±0.064, O.D 0.215±0.047 in comparison to O.D1.232±0.212, 1.597±0.225, respectively, in the presence of peptide D,p<0.001).

Inhibition of ICAM-1 expression was most impressive when G-3 mAb waspreincubated with peptide C and added to HUVEC (O.D 0.186±0.062 incomparison to OD 1.315±0.117, p<0.001, in the presence of peptide D),compared to ICAM-1 expression following exposure of HUVEC to peptide Aand ILA-1mAb (O.D. 0.204±0.072 compared to O.D 0.834±0.056 in thepresence of peptide D, p<0.002). The comparison between the inhibitoryeffect of peptides B and C on the adhesion molecules expression by HUVECcaused by ILA-3 and G-3 mAbs, respectively, was non-significant, p>0.05.

Inhibition of VCAM-1 expression showed the most significant effect withpeptide A and ILA-1 mAb (O.D 0.252±0.071 compared to O.D 1.372±0.203with the cocktail of irrelevant peptides B+C+D, p<0.001). G-3 mAb, whichhad less pronounced ability to activate VCAM-1 expression on HUVEC (O.D0.809±0.063 incubation with the cocktail of irrelevant peptides A+B+D,was inhibited by peptide C(O.D 0.174±0.062, p<0.004).

Example 5

Prevention of APS Induction in Naive Pregnant Mice by Peptides Specificto anti-β2GPI mAbs.

Significant fetal loss, thrombocytopenia and prolonged activatedthromboplastin time (aPTT) were induced in naive mice following passiveintravenous administration of the three anti-β2GPI mAbs ILA-1, ILA-3 andG-3, as described in Materials and Methods, section (j). The clinicalmanifestations of the experimental APS were prevented by infusion of thespecific peptide monomers A, B, C which were delivered to the mice withthe corresponding pathogenic anti-β2GPI mAbs, as shown in Table 2. Thecocktail of irrelevant peptides in each case did not affect thedevelopment of the disease symptoms. Similarly, mice which werepreinfused with control IgM and received a cocktail of the peptidemonomers (A+B+C), were not affected.

BALB/c mice which were preinfused with anti-β2GPI mAb ILA-1, and treatedthereafter with peptide monomer A, showed normal values of fetal loss(8±2%) compared to mice which were infused with the irrelevant peptidemonomer cocktail (B+C+D) (45±2%), or to non-treated mice (39±3%,p<0.001; p>0.5) when compared to mice infused with control human IgMthat was administered with the peptide cocktail (B+C+D). The plateletcount in the peptide A-treated mice was 989±103cells/mm³×10³, which isnormal in comparison to mice infused with the human IgM and treated withthe peptide cocktail (A+B+C), p>0.5. Significancy (p<0.003) was observedwhen the above platelet count was compared to non-treated ILA-1 infusedmice (498±142 cells/mm³×10³), or those treated with the peptide cocktail(B+C+D) (532±162 cells/mm³×10³). No prolongation in activated partialthromboplastin time (aPTT) was observed in mice infused with ILA-1 andtreated with peptide A (33±4 sec) compared to IgM-infused mice with andwithout treatment with the peptide cocktail (B+C+D) (31±2, 28±3 sec),p>0.05.

The same pattern of preventing effects by the specific peptides wasshown in mice infused either with pathogenic anti-β2GPI ILA-3 mAb andtreated with peptide monomer B or with pathogenic G-3 mAb and treatedwith peptide monomer C (Table 2). The values of fetal loss weresignificantly normal, p>0.5, in comparison to IgM-infused mice with andwithout treatment with the peptide cocktail; p<0.001 when compared toILA-3 or G-3 infused mice treated with the peptide monomer cocktail(A+C+D) or (A+B+D), respectively. No thrombocytopenia was detected inmice infused either with pathogenic anti-β2GPI ILA-3 mAb and treatedwith peptide B or with G-3 mAb and treated with peptide C, p>0.5 incomparison to IgM-infused mice with and without treatment with thepeptide cocktail (A+C+D) or (A+B+D), p<0.002 and p<0.001, respectively,when compared to ILA-3 or G-3 infused mice treated with peptide cocktail(A+C+D) or (A+B+D), respectively. Activated partial thromboplastin time(aPTT) in mice infused with either ILA-3 mAb and treated with peptide Bor G-3 mAb and treated with peptide C, was normal (27±2 and 31±4 sec,respectively), p>0.5 in comparison to IgM-infused mice with and withouttreatment with the peptide cocktail; p<0.002 when compared to ILA-3 orG-3 infused mice treated with peptide cocktail (A+C+D) or (A+B+D),respectively.

TABLE 2 Clinical manifestations in mice infused with anti-β2GPI mAbs andpeptides A, B, C Infusion of mAb: ILA-1 ILA-3 G-3 HlgM Peptide: A NONB + C + D B NON B + C + D C NON B + C + D NON A + B + C aPTT(SEC.) @ 33± 4  72 ± 5 78 ± 3 27 ± 2 64 ± 3 69 ± 3 31 ± 4 83 ± 5 79 ± 4 31 ± 2 28 ±3 (p < (p < (p < 0.02)* 0.02)* 0.02)* Platelet count @ 989 ± 498 ± 532 ±1137 ± 579 ± 601 ± 1242 ± 499 ± 676 ± 1189 ± 1207 ± (cells/mm³ × 10⁻³)103 142 162 219 163 134 267 112 105 273 212 (p < (p < (p < 0.003)*0.002)* 0.001)* % Fetal loss 8 ± 2 39 ± 3 45 ± 2  6 ± 2 42 ± 3 49 ± 4  7± 2 42 ± 4 39 ± 4  6 ± 2  8 ± 4 (p < (p < (p < 0.001)* 0.001)* 0.001)*Values are expressed as mean ± SD of 2 experiments; N = 11–15 mice ineach group. @ aPTT: activated partial thromboplastin time. % Feal loss =Resorbed fetuses/Total fetuses *Statistical analyses were performed byANOVA test. Groups of mice treated with peptide A, B or C, were comparedto groups of mice treated with cocktail of peptides (B + C + D), (A +C + D) or (A + B + D) respectively.

Example 6

Frequency of anti-β2GPI Target Epitopes A, B and C in Patients withPrimary APS or Secondary to SLE

Binding of sera from patients with primary APS or secondary to SLE tothe studied peptides was performed by ELISA. 96-well ELISA plates werecoated with streptavidin, blocked with gelatin, incubated withbiotinylated peptides (A,B,C,D) and blocked with gelatin. Sera ofpatients with APS or SLE were added at dilution of 1:50 and the percentof binding was probed with anti-human IgM or IgG conjugated to alkalinephosphatase. Half plate was coated with streptavidin without addition ofpeptide (for non-specific binding).

FIG. 5 shows the direct binding of anti-β2GPI IgM and IgG of APSpatients to peptides A, B, C, and D, presented in O.D. at 405 nm. Thesera from the APS patients were found to bind differentially the testedpeptides, either as a result of distinct affinity to the peptides or dueto different titers of antibodies recognizing the target epitopes.

As shown in Table 3, peptide A was recognized by anti-β2GPI IgM andanti-β2GPI IgG in 21% and 7%, respectively, out of 43 APS patients, andin 11% and 5.5%, respectively, out of 72 SLE patients. Peptide B wasrecognized by anti-β2GPI IgM and anti-β2GPI IgG in 25.6% and 20.9%,respectively, out of 43 APS patients, and in 12.5% and 5.5%,respectively, out of 72 SLE patients. Peptide C was recognized byanti-β2GPI IgM and IgG in 32.5% and 16.3%, respectively, out of 43 APSpatients, and in 15.3% and 6.8%, respectively, out of 72 SLE patients.

As shown in Table 4, the percentage of antibodies from the totalanti-β2GPI affinity purified Abs (from 25 APS patients) recognizing theinhibitory monomer peptides A, B C, was in the range between 44% to0.5%.

TABLE 3 Percent of sera from APS and SLE recognizing the inhibitorypeptide A, B and C APS patients SLE patients Normal donors (N = 43) (N =72) (N = 100) IgG IgM IgG IgM IgG IgM Peptide A 7 20.9 5.5 11 0 2Peptide B 20.9 25.6 11 12.5 0 1 Peptide C 16.3 27.9 2.8 14 1 0 Peptide D0 0 0 0 2 1 N = Number of patients studied

TABLE 4 Fraction of anti-β2GPI Abs (in percentage), recognizing peptideA, B, or C, in each APS patient studied Patient no. PEPTIDE A PEPTIDE BPEPTIDE C 1 23 37 0.4 2 0.7 15 3 3 1.4 0.5 39 4 29 43 22 5 0.9 29 1.8 629 14 23 7 37 18 5 8 2.8 33 4.2 9 12 1.3 0.5 10 0.6 7.9 16.4 Affinitypurified anti-β2GPI Abs were loaded on peptide A column, eluted andpassed through peptide B column followed by peptide C column. Thepercentage of anti-peptide A, B, and anti-peptide C were calculated fromtotal anti-β2GPI Abs.

Example 7

Specificity of Recognition of the anti-β2GPI Epitopes in Primary APSPatients

The specificity of the recognition of the studied anti-β2GPI epitopes A,B, C by anti-β2GPI Abs from APS patients was confirmed by competitionassays in which specificity of binding of anti-β2GPI IgM and IgG frompatients with APS to the inhibitory peptides was examined. The resultsare shown in FIGS. 6A–6C. Anti-β2GPI IgG and IgM affinity purified fromAPS patients (affinity purification of the antibodies was carried out byincubation of cardiolipin lyposomes with patients' sera overnight at 4°C. with rotation following sedimentation of the complexes (30000 rpm),elution of the bound Abs by KI 1M, extraction from the lyposomes bychloroform, and separation of the total anti-β2GPI IgM, IgG Abs into thedifferent isotypes employing either anti-human-IgM-Sepharose oranti-human-IgG-Sepharose (Pharmacia)) were preincubated with β2GPI(triangles), specific peptide monomer (circles) or a cocktail of theother peptides as monomers (squares), and tested for their binding tothe specific biotinylated peptide-coated ELISA plates. Anti-β2GPI IgMand IgG from APS patients recognized specifically ILA-1 correspondingpeptide A, as shown in FIG. 6A, by competition with peptide A incomparison to peptide cocktail (B+C+D) as competitors (p<0.002). β2GPIelicits the anti-β2GPI IgM and IgG binding to peptide A, leading to thepostulation that epitope A is exposed on the β2GPI molecule. On theother hand, anti-β2GPI IgM and IgG which were found to bind specificallypeptide B (FIG. 6B), did not recognize the β2GPI molecule in fluidphase, since β2GPI could not abrogate the binding of anti-β2GPI topeptide B (p>0.5), pointing to the possibility that peptide B is acryptic epitope of the β2GPI molecule. Peptide C, corresponding to H-3anti-β2GPI mAb, was recognized specifically by anti-β2GPI IgM and IgGfrom APS patients (p<0.002). β2GPI molecule was able to reduce theanti-β2GPI binding to peptide C in fluid phase, pointing to thepossibility that this epitope is exposed on the surface of the targetmolecule (FIG. 6C).

Example 8

The Effect of the Inhibitory Peptides A, B, C on anti-β2GPI Secretion byHybridoma Cells

Anti-β2GPI mAb secretion by human hybridoma cells named ILA-1, ILA-3,G-3 or human hybridoma secreting irrelevant immunoglobulin PE wasstudied by ELISA. The hybridoma cells were incubated in vitro with thepeptides A, B, C or D as monomers, dimers or tetramers with St. The PEhybridoma cells were exposed to a cocktail of tetramer peptidesSt-tetraA+St-tetraB+St-tetraC+St-tetraD. The peptides were used at aconcentration of 10 μM.

The results shown in FIG. 7 reveal that addition of St-tetraA to ILA-1cells, St-tetraB to ILA-3 cells and St-tetraC to G-3 cells completelyabrogated the antibody secretion (p<0.001), while irrelevant St-tetraDhad no effect. Addition of a cocktail of the 4 tetramer peptides toirrelevant PE hybridoma cells had no effect on the Ab secretion.Administration of the specific dimer peptides A, B. C to thecorresponding hybridoma cells abolished moderately, but significantly,the anti-β2GPI Ab secretion (p<0.03).

Example 9

Effect of the Inhibitory Peptides on Human anti-β2GPI Antibody Secretion

The effect of the inhibitory peptides A, B, C as tetravalent MAP withLys on anti-β2GPI Abs secretion by PBL from APS patients was tested invitro as described in Materials and Methods, section (1), with PBLderived from APS patient S, who was the source for the preparation ofILA-1 and ILA-3 mAbs, and from APS patient Y, whose anti-β2GPI Abrecognized peptide A. PBL from a human donor that does not produceanti-β2GPI Abs were used as control. The tetravalent peptides A, B, A+B,C, and D were used to test patient S, and A, B, and C to test patient Y.The results are shown in FIG. 8.

As shown in FIG. 8, the tetravalent peptides A and B inhibited specificanti-β2GPI Ab secretion by PBLs derived from patient S. IgM secretionwas reduced to O.D 0.378±0.047, IgG secretion was reduced to O.D0.625±0.037 in the presence of the tetravalent peptide A, in comparisonto anti-β2GPI IgM and IgG secretion in the presence of the irrelevanttetravalent peptide D (O.D 0.827±0.063 and 0.934±0.083, p<0.001 andp<0.04, respectively). The tetravalent peptide A reduced the secretionof anti-β2GPI IgM (p<0.002) and of IgG (p<0.03) by PBL derived from APSpatient S. The tetravalent peptide B inhibited significantly theanti-β2GPI IgM secretion (p<0.002) and IgG secretion (p<0.04) by PBLfrom APS patient S, but not by PBL originated from APS patient Y, p>0.5.The most pronounced inhibitory effect on anti-β2GPI IgM and IgGsecretion was shown when a mixture of tetravalent peptides A+B was givento patient S (p<0.001 and p<0.002, respectively).

Example 10

Effect of the Inhibitory Peptides as Monomers, Divalent and TetravalentMAP on anti-β2GPI Antibody Forming Cell Activity

Enriched population of anti-β2GPI B cells was studied for anti-β2GPI IgMand IgG AFC activity in the presence tetravalent inhibitory peptides A,B, A+B, and control D, by spot ELISA, as described in Materials andMethods, section (1). The results in FIG. 9 show significant abrogationin the number of IgM anti-β2GPI AFCs upon exposure to tetravalentpeptide A, (37%), and inhibition of 40.5% of IgG anti-β2GPI AFCs(p<0.002). Exposure of the anti-β2GPI B cells to tetravalent peptide Bresulted in significant reduction in the number of IgM anti-β2GPI AFCsby 59% (p<0.001) and 21.7% inhibition of anti-β2GPI IgG secretion(p<0.02) was observed. Synergistic effect was shown when the studiedcells were treated with a mixture of tetravalent peptides A+B, thatinhibited IgG anti-β2GPI AFCs by 47% and IgM by 82%, pointing to thepossibility that there are additional epitope/s recognized by anti-β2GPIIgG. Tetravalent peptide C (anti-β2GPI corresponding epitope which isnot recognized by the immunoglobulins from the studied patient S), andirrelevant tetravalent peptide D did not affect anti-β2GPI AFC activity(2–10%) p>0.5.

Dose dependent studies on human anti-β2GPI AFC activity were carried outwith enriched B cell population from an APS patient, specific to β2GPI,exposed to varying concentrations of the inhibitory peptides A, B, C asmonomers, divalent and tetravalent peptides, and to irrelevant anti-DNAAFC as negative control. The results are shown in FIGS. 10A–C. FIG. 10A:monomer A, divalent peptide A, tetravalent peptide A and control peptideD. FIG. 10B: monomer B, divalent peptide B, tetravalent peptide B andcontrol peptide D. FIG. 10C: FIG. 10C: monomer mixture A+B, divalentpeptide mixture A+B, tetravalent peptide mixture A+B and control peptideD The results confirm the most significant inhibitory effect of thespecific tetravalent peptide on antibody-forming cell activity (p<0.001for all specific peptides and p>0.05 for irrelevant peptide).

Example 11

Detection of B Cell Epitopes in Mice

BALB/c mice were immunized (day 0) in the hindfootpads with 10 μg β2GPIin Complete Freund's Adjuvant (CFA), Three weeks after, the micereceived a booster injection of β2GPI in PBS. Sera of the mice(anti-β2GPI) were used to screen peptides for the presence of B cellepitopes, as follows: 96-well ELISA plates were coated with streptavidinovernight at 4° C., followed by incubation with biotinylated peptidemonomers A, B, C, or D, β2GPI or BSA in PBS (100 μM) for 2 hours at roomtemperature. The ELISA plates were blocked with 3% gelatin, and themouse sera were added thereto at different dilutions. Binding was probedwith goat anti-mouse IgG alkaline phosphatase. The results depicted inFIG. 11 show that peptides A, B and C reacted specifically withantibodies from the immunized mice (p<0.001 for peptides A,B,C comparedto peptide D), indicating the presence of B cell epitopes on the threepeptides.

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1. A synthetic peptide selected from the group consisting: (i) a peptideof SEQ ID NO:1 to SEQ ID NO: 7, of the sequences:Leu-Lys-Thr-Pro-Arg-Val (SEQ ID NO:1); Lys-Thr-Pro-Arg-Val-Thr (SEQ IDNO:2); Asn-Thr-Leu-Lys-Thr-Pro-Arg-Val-Gly-Gly (SEQ ID NO: 3);Lys-Asp-Lys-Ala-Thr-Phe-Gly-Thr-His-Asp-Gly (SEQ ID NO:4);Thr-Lys-Leu-Arg-Val-Tyr (SEQ ID NO:5); Thr-Leu-Leu-Arg-Val-Tyr (SEQ IDNO:6); and Cys-Ala-Thr-Leu-Arg-Val-Tyr-Lys-Gly-Gly (SEQ ID NO:7), and(ii) a peptide of (i) that is biotinylated.
 2. The peptide according toclaim 1 of SEQ ID NO:1.
 3. The peptide according to claim 1 of SEQ IDNO:4.
 4. The peptide according to claim 1 of SEQ ID NO:7.
 5. The peptideaccording to claim 1 (ii) that is biotinylated.
 6. The peptide accordingto claim 5, wherein the peptide is a peptide of SEQ ID NO:1 that isbiotinylated.
 7. The peptide according to claim 5, wherein the peptideis a peptide of SEQ ID NO:4 that is biotinylated.
 8. The peptideaccording to claim 5, wherein the peptide is a peptide of SEQ ID NO:7that is biotinylated.